Vitamin D3 (Vit D) and its metabolites have been receiving increasing attention in health-related studies due to their role in numerous cellular functions, including cell differentiation and matrix turnover. The primary route for Vit D synthesis in the human body is through UV-B irradiation of the skin via sunlight. We propose to study Vit D synthesis and metabolism in the cornea, one of the few other tissues that receives direct UV-B irradiation. We hypothesize that Vit D and Vit D metabolites are present in the cornea and anterior chamber, and that they can be both synthesized in the cornea and can enter the anterior chamber via the circulation. We also hypothesize that Vit D metabolites can influence corneal epithelial cell differentiation. Preliminary data supports these hypotheses. The Specific Aims of this R-21 application are designed to test these hypotheses, and include: (1) Determine if the presence of Vit D and 1,25-(OH)2D in the aqueous humor are influenced by dietary Vit D intake and/or UV exposure;(2) Localize the source(s) of 11-hydroxylase in the cornea;and (3) Determine if 1,25-(OH)2D activity is important for epithelial wound coverage and re-differentiation during corneal wound healing. Animal studies will be performed utilizing knockout mouse models as well as dietary Vit D reduction and supplementation to explore corneal Vit D metabolism and cell differentiation. Cell culture studies will be employed to study Vit D synthesis by corneal cells, as well as the influence of Vit D metabolites on epithelial barrier function. The long term goal of this work is to lay a foundation for future studies of Vit D in the eye. We plan to follow up this work with an R01 application to carry out this future Vit D research. PUBLIC HEALTH RELEVANCE Recent work suggests that more than 50% of the US population suffers from Vitamin D (Vit D) insufficiency. Of the tens of thousands of publications examining Vit D, only a handful has examined Vit D in the cornea and anterior segment, and none of these looked at actual Vit D concentrations, metabolism, or activity. By measuring these parameters in the cornea and anterior segment of the eye, along with how Vit D affects cell differentiation in this tissue, this work set the foundation for future Vit D work in the eye.